The advent of sophisticated molecular biology techniques has conferred a greater understanding of the mechanisms of diseases, which has led to the design and synthesis of drugs that act at specific sites within cells. One example is antisense oligonucleotides, which must bind to specific target mRNAs in the cytoplasm or nucleus of diseased cells. See Lebedeva et al, 2000, Eur. J. Pharm. Biopharm. 50(1)101–119 and Crooke, 1999, Biochem. Biophys. Acta 1489(1)31–44. Delivery of the drug to the target site is paramount, as even the most potent drug will be ineffective if it cannot interact with its target. The simplest path by which drugs enter cells is diffusion. It has long been accepted that diffusion through a plasma or organelle membrane is restricted to small molecules, which is a major reason why most therapeutics are small molecules. See Foster et al, 1988, Biochim. Biophys. Acta 947(3)465–491. Thus, the requirement for the delivery of drugs to specific sites is contrary to the nature of high-molecular-weight (HMW) drugs and polymer carriers. However, even small diffusible drugs may not affect their target sites within cells. Disadvantages of drugs entering cells by diffusion include a requirement of high permeability and possible efflux (e.g., multidrug resistance due to the efflux of drugs by membrane pumps such as P-glycoprotein). See Kipecek, et al, 2000, Eur. J. Phar. Biophar. 50(1)61–81; Ryser et al, 1978, Proc. Natl. Acad. Sci. 75(4)3867–3870; and Ohkawa et al, 1993, Cancer Res. 53(18)4238–4242.
The large size and potential charge of HMW compounds prevents them from entering as many compartments as small molecules. See de Duve et al, 1974, Biochem. Pharmacol. 23(18)2495–2531. The biodistribution of small molecules is principally governed by their permeability and affinity for biological components with diffusion being their typical method of entry into biological compartments. If the desired balance in solubility and permeability of small drug molecules can be obtained by chemical modification, the drug should be able to reach most compartments and interact with its target. In contrast to small molecules, HMW material is internalized by endocytosis. Adding ligands to macromolecules can target the compound to specific cells, and thereby result in increased uptake, but once the material has been endocytosed, it still remains separated from the cell's interior by a biological membrane.
The most common fate of endocytosed material is delivery to the lysosome, where high levels of lysosomal enzymes are present. Drugs sensitive to these enzymes will be quickly degraded if steps are not taken to protect them or to facilitate their escape into the cytosol. The limited number of compartments accessible to macromolecules and probably exposure to a lower pH and degradative enzymes decreases their probability of success. Delivery of these and other HMW compounds to their target site is currently one of the greatest obstacles for their success. Since many drugs today are being designed to work at specific sites within cells, it would be very desirable to improve their delivery to the desired subcellular compartment. See Jensen et al, J. Controlled Release 87(2003)89–105.
The contractile apparatus in muscle cells, known as the sarcomere, is surrounded by a specialized membrane-bound sub-cellular structure called the sarcoplasmic reticulum (SR). There is a fundamental need to better understand SR protein interactions as well as develop therapies that act directly on SR-resident proteins. Therefore, a means is needed for targeting molecules to the SR as either a research tool or a therapeutic delivery system. One potential solution would be to use a component of an SR-resident protein as a targeting signal.
Intracellular calcium pools are maintained within the SR. Gradients of intracellular calcium control diverse biological activities, including muscle contraction. A class of proteins termed Sarco(endo)plasmic recticulum Calcium ATPases (SERCAs) are responsible for pumping calcium into the SR. Alterations in SERCA activity are associated with numerous disease states, including Darier disease and cardiac failure. Alterations in SERCA activity can be caused by changes in transcriptional or post-transcriptional regulation of SERCA gene expression, biochemical modification of a SERCA protein, and/or biophysical interactions of a SERCA protein with other proteins.
The SR-localized protein phospholamban (PLN) interacts with SERCA via biophysical associations. PLN is a 52-amino acid polypeptide with three domains, known as Ia, Ib, and II (Fujii et al, 1987, J. Clin. Invest. 79, 301–304). Conservation of PLN polypeptide sequence between mouse and human genes is 100%. There is a single amino acid substitution at amino acid 2 in canine and porcine compared to murine and human. PLN domain Ia (amino acid 1–20) is highly charged and helical, domain Ib (amino acid 21–30) is polar and unstructured, while domain II (amino acid 31–52) is neutrally-charged, very hydrophobic, and assumes an alpha-helical structure in a physiological milieu (Mortishire-Smith et al, 1995, Biochem. 34, 7603–7613). Further studies of domain II showed that the alpha-helix forms a transmembrarie domain that spans the lipid bilayer membrane of the SR. Biologically active PLN is a homopentamer of 5 PLN polypeptides. Experiments in cultured cells showed that the transmembrane domain of PLN associates with SERCA and inhibits calcium uptake into the SR (Kimura et al, 1996, J. Biol. Chem. 271, 21726–21731).
Further work studied mutations in individual amino acids in the PLN transmembrane domain to determine their effects on SERCA function (Kimura et al, 1997, J. Biol. Chem. 272, 15061–15064). In these studies, 22 different expression constructs were made with a single amino acid mutation of the transmembrane domain in each, so that each amino acid was changed to alanine in one of the constructs. Each mutant construct was transfected into mammalian cells, along with expression constructs for a SERCA protein, and SERCA function was assayed in each. While 14 of the mutations either maintained or increased inhibition of SERCA, 8 of the mutations abolished the ability of the PLN transmembrane domain to inhibit SERCA-driven calcium pumping.
While Kimura demonstrated that a single mutation to cause substitution of alanine for L31, N34, F35, 138, L42, 148, V49, or L52 (amino acid residues within the polypeptide of the transmembrane domain) disrupts inhibition of SERCA function in vitro, there has been no report of any successful application of this concept to conduct a protein to the SR. The nature of biological systems makes it difficult to predict accurately whether a mutated PLN transmembrane domain would in fact allow targeting of compounds or macromolecules to the SR. Thus, it remains unclear whether a mutant PLN transmembrane domain can serve as an SR targeting signal for a therapeutic or experimental compound or macromolecule without interferring with normal cellular functions, especially those associated with the SR.